Project Summary The human gastric pathogen Helicobacter pylori infects half the world?s population and is remarkably persistent, able to avoid clearance by the immune system for the duration of an individual?s lifetime. Chronic infection by the bacterial pathogen is now known to be the major cause of peptic ulcers and is associated with the development of adenocarcinoma. A simple chemotaxis system allows H. pylori to survive in the harsh environment of the stomach by navigating toward beneficial chemicals and away from harmful ones, yet the mechanism of this process is poorly understood. Emerging evidence has unexpectedly revealed that chemotaxis by a cytosolic receptor TlpD plays an important role in H. pylori colonization. Most previous work has focused on membrane-bound chemoreceptors, even though cytosolic chemoreceptors are common amongst prokaryotes. The cytosolic receptor TlpD has been shown to sense reactive oxygen species (ROS) and is essential for H. pylori to colonize and grow normally in the stomach antrum. We hypothesize H. pylori uses its cytosolic chemoreceptor TlpD to sense ROS excreted by immune cells and that this allows it to avoid killing by host macrophage and neutrophils. We propose to test this hypothesis with a combined molecular-level and cellular- level approach. Specific Aims: (1) Test the hypothesis that hydrogen peroxide induces a structural change in TlpD that facilitates signal transduction. (2) Test the hypothesis that ROS-sensing by TlpD provides a survivorship advantage by detecting and evading host immune cells. Research Design: To understand the function of TlpD, we will use biophysical approaches like fluorescence anisotropy, analytical ultracentrifugation, and protein crystallography to examine the interactions of the receptor with other chemotaxis proteins, and determine the molecular mechanism by which the receptor senses ROS and transmits this signal to chemotaxis kinases. We will apply this knowledge to understand the pathogen?s behavior in vivo by engineering mutant H. pylori strains and testing their ability to sense ROS. I will assess whether immune cells produce a chemical signal detectable by H. pylori, and if TlpD confers a survivorship advantage by facilitating chemorepulsion from neutrophils and macrophage. This research will improve understanding of the biologically and medically important process of chemotaxis for H. pylori, and lay the foundation to target elements of the chemotaxis system with novel therapeutics. Our broad long term goal is to advance the field of bacterial chemotaxis by elucidating the mechanism by which a cytosolic receptor can sense an extracellular signal.